4-Chlorobenzoate:coenzyme A (4-CBA:CoA) ligase catalyzes 4-chlorobenzoyl-coenzyme A formation in a two-step reaction consisting of the adenylation of 4-chlorobenzoate with adenosine 5'-triphosphate followed by acyl transfer from the 4-chlorobenzoyl adenosine 5'-monophosphate diester intermediate to coenzyme A. In this study, two core motifs present in the Pseudomonas sp. strain CBS3 4-CBA:CoA ligase (motif I, 161T-S-G-T-T-G-L-P-K-G170, and motif II, 302Y-G-T-T-E306) and conserved among the sequences representing the acyl-adenylate/thioester-forming enzyme family (to which the ligase belongs) were tested for their possible role in substrate binding and/or catalysis. The site-directed mutants G163I, G166I, P168A, K169M, and E306Q were prepared and then subjected to steady-state and transient kinetic studies. The results, which indicate reduced catalysis of the adenylation of 4-chlorobenzoate in the mutant enzymes, are interpreted within the context of the three-dimensional structure of the acyl-adenylate/thioester-forming enzyme family member, firefly luciferase.

Microbial enzyme systems may be used in the biodegradation of persistent environmental pollutants. The three polypeptide components of one such system, the 4-chlorobenzoate dehalogenase system, have been isolated, and the chemical steps of the 4-hydroxybenzoate-forming reaction that they catalyze have been identified. The genes contained within a 4.5-kilobase Pseudomonas sp. strain CBS3 chromosomal DNA fragment that encode dehalogenase activity were selectively expressed in transformed Escherichia coli. Oligonucleotide sequencing revealed a stretch of homology between the 57-kilodalton (kD) polypeptide and several magnesium adenosine triphosphate (MgATP)-cleaving enzymes that allowed MgATP and coenzyme A (CoA) to be identified as the dehalogenase cosubstrate and cofactor, respectively. The dehalogenase activity arises from two components, a 4-chlorobenzoate:CoA ligase-dehalogenase (an alpha beta dimer of the 57- and 30-kD polypeptides) and a thioesterase (the 16-kD polypeptide).

# These authors contributed equally to this work. Keywordsepigenetics; histone demethylase; bipyridyl; enzyme inhibitors; 2-oxoglutarate In eukaryotes, nuclear DNA is packaged into chromatin by binding to histones and associated factors. Covalent modifications to histone tails are associated with specific transcriptional states of the associated DNA. Acetylation of lysine side-chains normally correlates with transcriptional activation, while deacetylation leads to transcriptional silencing. The regulatory roles of methylation of lysine and arginine residues appear to be more complex. Methylation of certain lysine residues is associated with active transcription, while methylation of others is associated with silencing and heterochromatin formation. Each methylation 'mark' is placed, removed and 'interpreted' in a site-specific manner by histone methyltransferases, demethylases and methyl-binding domains, respectively. The biological functions of the individual enzymes are largely undefined, and are the focus of current investigations (for review see [1,2] The JmjC histone demethylases are 2-oxoglutarate (2OG) dependent oxygenases that catalyse N ε -lysyl demethylation via hydroxylation of the methyl group in a 2-oxoglutarate and Fe(II)-dependent manner (Scheme 1). Human 2OG oxygenases catalyse a range of reactions, including hydroxylation of amino acids, DNA and small molecules, and demethylation of proteins and DNA. [3] 2OG oxygenases show promise as therapeutic targets. An inhibitor of γ-butyrobetaine hydroxylase (BBOX) is used for the treatment of cardiovascular disease [4,5] and inhibitors of the hypoxia inducible factor (HIF) prolyl hydroxylases are in clinical trials for the treatment of anaemia. [6] Inhibitors of the collagen prolyl hydroxylases have also been evaluated as potential therapeutics for the treatment of liver fibrosis. [7,8] The discovery of the JmjC domain histone demethylases, and the suggestions that some of them are potential therapeutic targets for cancer treatment, [9] Figure 1). [10][11][12][13] Compounds which catalyse the ejection of a structural Zn(II) ion from the JMJD2 demethylases have also been reported ( Figure 1). [14] In a study describing various template inhibitors of the JmjC demethylases, we found that 2,2′-bipyridyl compounds with at least one 4-carboxylate group inhibit the histone demethylase JMJD2E. [11] A related series of compounds, 5,5′-dicarboxylate-2,2′-bipyridyls, is reported to inhibit the collagen prolyl-4-hydroxylases. [15] 2,2′-Bipyridine and bipyridyl compounds have also been used as inhibitors of the HIF hydroxylases. [16] Although it is likely that in some cases the enzyme inhibition effects of bipyridyl compounds result from metal chelation in solution, they also have the potential to inhibit via active site binding, as is the case for some 2OG oxygenases; however, to date there is no structural information on their mechanism of action. Here we report structure-activity relationship studies and analyses on bipyridyl inhibitors of JMJD2E.The bipyridyl com...

4-Chlorobenzoate:coenzyme A ligase (4-CBA:CoA ligase) catalyzes the first step of the 4-CBA degradation pathway of Pseudomonas sp. strain CBS3. In this reaction, 4-CBA-CoA thioester synthesis is coupled to ATP cleavage. The studies described in this paper examine the intermediacy of 4-chlorobenzoyl-adenosine 5'phosphate diester (4-CBA-AMP) in the ligase reaction. The 4-CBA-AMP adduct was isolated from the ligase reaction mixture generated from magnesium adenosine 5-triphosphate (MgATP) and 4-CBA in the absence of CoA. The structure of the 4-CBA-AMP was verified by 1H- 13C-, and 31P-nuclear magnetic resonance analysis. Single-turnover reactions carried out with 14C-labeled 4-CBA in a rapid quench apparatus demonstrated formation of the enzyme. 4-CBA-AMP.MgPPi complex from the enzyme.4-CBA.MgATP complex at a rate of 135 s-1. The rate of ligand release from the enzyme.4-CBA-AMP.MgPPi complex was measured at 0.013 s-1. Single-turnover reactions of [14C]-4-CBA, MgATP, and CoA catalyzed by the ligase revealed that the 4-CBA-AMP intermediate formed reaches a maximum level of 25% of the starting 4-CBA within 10 ms and then declines with the formation of the 4-CBA-CoA. The rates of the adenylation and thioesterification partial reactions, determined by kinetic simulation of the rate data, are nearly equal (135 and 100 s-1). Substitution of CoA with the slow substrate pantetheine did not significantly alter the rate of the adenylation step but did reduce the rate of the thioesterification step to 2 s-1. The maximum level of 4-CBA-AMP reached during the single-turnover reaction of 4-CBA, MgATP, and pantetheine corresponded to one-half of the starting 4-CBA.

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